ABSTRACT
Cleavage after lysine 32 in the Ggamma2 subtype and after lysine 36 in the Ggamma3 subtype of purified mixed brain Gbetagamma by endoproteinase Lys-C blocks Gbetagamma-mediated stimulation of phosphorylation of rhodopsin in urea-extracted rod outer segments by recombinant human beta-adrenergic receptor kinase (hbetaARK1) holoenzyme while hbetaARK1 binding to rod outer segments is partially affected. This treatment does not attenuate the binding of the treated Gbetagamma to C-terminal fragments of hbetaARK1 containing the pleckstrin homology domain. Lys-C proteolysis also does not alter the association of the Gbetagamma with phospholipids, its ability to support pertussis toxin-catalyzed Galphao/Galphai ADP-ribosylation, or its ability to inhibit forskolin-stimulated platelet adenylate cyclase. The Gbeta subunit remains noncovalently associated with the cleaved Ggamma fragments. Thus, in addition to recruiting hbetaARK1 to its receptor substrate, Ggamma contributes secondary and/or tertiary structural features to activate the kinase.
Subject(s)
Brain/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , GTP-Binding Proteins/metabolism , Rhodopsin/metabolism , Adenylyl Cyclase Inhibitors , Adenylyl Cyclases/blood , Amino Acid Sequence , Animals , Binding Sites , Blood Platelets/enzymology , Cattle , Chromatography, High Pressure Liquid , Enzyme-Linked Immunosorbent Assay , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/isolation & purification , Humans , Kinetics , Lysine , Macromolecular Substances , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Phospholipids/metabolism , Phosphorylation , Recombinant Proteins/metabolism , Rod Cell Outer Segment/metabolism , beta-Adrenergic Receptor KinasesABSTRACT
Receptor phosphorylation is a key step in the process of rapid desensitization. beta-adrenergic receptor kinase (betaARK) is a specific receptor kinase that is known to phosphorylate and induce desensitization of several G-coupled receptors only when they are occupied by their agonists. In the present study we have done several modifications to the amino-terminal of betaARK1, in order to clarify its functional role. The recombinant mutants were tested for their ability to phosphorylate rhodopsin present in purified bovine ROS membranes which serves as a substrate for betaARK1. Their expression levels were detected by Western blot analysis. We found that when the amino-terminal of betaARK1 is modified its expression level is very low, hence it is not able to phosphorylate over the basal. These findings suggest that this region is crucial for the normal processing of the protein.
